RESUMO
Excision repair cross-complementing 1-118 single nucleotide polymorphisms (SNPs) have been reported as predictive markers of response to platinum-based chemotherapy. Currently, the most used methods for genotyping of SNPs are costly and time consuming. It is necessary to develop a method that is more accurate, cost-effective, and simple. An improved fluorescence polarization assay based on asymmetric polymerase chain reaction hybridization for screening excision repair cross-complementing 1-118 SNPs has been developed. Excision repair cross-complementing 1-118 SNPs of all 907 samples were analyzed in sequence and improved fluorescence polarization assay in parallel. The sensitivity, specificity, and stability of the improved fluorescence polarization assay were measured. This study showed the accuracy, simplicity, and cost-effectiveness of the fluorescence polarization assay in the detection of excision repair cross-complementing 1-118 SNPs in a panel of 907 samples. The fluorescence polarization assay was more accurate for the heterozygous sample than was the sequence assay. The minimum detection level established with the fluorescence polarization assay was 2.5 genome copies per reaction and no cross-reaction was observed.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Polarização de Fluorescência/métodos , Patologia Molecular/métodos , Polimorfismo Genético , Humanos , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
OBJECTIVES: Herpes simplex virus type 1/2 (HSV-1/-2), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) correlate strongly with infections of the central nervous system. The objective of this study was to develop a method for the simultaneous detection of HSV-1/-2, CMV, and EBV DNA by the fluorescence polarization assay based on asymmetric polymerase chain reaction (PCR) and hybridization. METHODS: DNA of HSV-1/-2, CMV, and EBV was amplified in an asymmetric PCR by a universal primer system. The amplicons were then detected by the fluorescence polarization assay. In this method, the probes for HSV-1/-2, CMV, and EBV hybridized with their respective target amplicons, and the hybridization resulted in an increase in the fluorescence polarization values. Infections of HSV-1/-2, CMV, and EBV were determined by the increased fluorescence polarization values. The DNA extracted from whole blood and cerebrospinal fluid samples was subjected to fluorescence polarization and a previously published multiplex PCR assay in parallel. RESULTS: Compared to the multiplex PCR assay, no significant difference in the numbers of samples positive for the human herpesviruses was identified by the fluorescence polarization assay. CONCLUSIONS: The fluorescence polarization assay presented in this study is a reliable, convenient, and cost-effective diagnostic tool that allows the detection of the four major human herpesviruses.
Assuntos
Polarização de Fluorescência/métodos , Infecções por Herpesviridae/diagnóstico , Herpesviridae/isolamento & purificação , DNA Viral/sangue , DNA Viral/líquido cefalorraquidiano , Infecções por Herpesviridae/sangue , Infecções por Herpesviridae/líquido cefalorraquidiano , Humanos , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
The hepatitis B virus genotypes A and D have global distributions, while the hepatitis B genotypes B and C are predominant in Asia. Individuals infected with genotype C or D have a lower response rate to therapy than individuals infected with genotype A or B. The conventional detections of hepatitis B genotypes A to D, however, have not been used routinely due to technical difficulties and high costs. A simple and cost-effective method for simultaneous detection of the hepatitis B genotypes A to D has been developed. A pair of general primers in the preS region of the hepatitis B were used in an asymmetric PCR. Four probes specific for the hepatitis B genotypes A to D labeled with different fluorophores hybridized, respectively with their target amplicons, and the hybridization increased the fluorescence polarization (FP) values. The genotypes were determined by the increased fluorescence polarization values. DNA extracted from 1398 samples was detected by the FP and the type-specific PCR assay in parallel. No significance difference was found between the two methods.
Assuntos
Polarização de Fluorescência/métodos , Vírus da Hepatite B/classificação , Vírus da Hepatite B/isolamento & purificação , Hepatite B/diagnóstico , Hepatite B/virologia , Reação em Cadeia da Polimerase/métodos , Virologia/métodos , Primers do DNA/genética , Fluorescência , Genótipo , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Sondas de Oligonucleotídeos/genética , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: The detection of a broad spectrum of HPV genotypes has not been used widely in cervical cancer screening due to technical difficulties and high costs. OBJECTIVES: To develop an asymmetric GP5+/6+ PCR assay and hybridization with a fluorescence polarization (FP) assay of 15 HPV genotypes. STUDY DESIGN: HPV genes in controls and samples were amplified by an asymmetric GP5+/6+ PCR. Fifteen HPV genotypic probes labeled with fluorophores hybridized with target PCR product to identify the presence of specific HPV genotypes. The HPV genotypes in samples were verified by sequence assay. RESULTS: The genotypes determined with the hybridization and FP assay were confirmed by sequence analysis when a monotypic infection was evaluated. CONCLUSION: A simple, economical and specific HPV genotyping assay has been developed that will be adequate for cervical cancer screening programs.
